Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer.
A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.
Nolta
J A
JA
Division of Research Immunology/Bone Marrow Transplantation, Children's Hospital of Los Angeles, CA 90054-0700.
Sender
L S
LS
Barranger
J A
JA
Kohn
D B
DB
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D001853:Bone Marrow / Q000201:enzymology*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005959:Glucosidases / Q000235:genetics*; D005962:Glucosylceramidase / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D013997:Time Factors; D014162:Transfection
D004247:DNA; D005959:Glucosidases; D005962:Glucosylceramidase